The primary objective of this project, an expansion of an ongoing productive collaboration, is to further develop and refine the use of phosphate analogues for the high-resolution study of protein-DNA interactions. This involves the development of new and improved methods for the stereospecific synthesis of chiral phosphate analogues (H- phosphonates, phosphorothioates and methanephosphonates) and the "calibration" of the effects of these analogues with a relatively well- characterized protein-DNA complex (Eco RI endonuclease). The concordance of results with the various analogues will permit us to probe protein interactions with individual phosphoryl oxygens in DNA at atomic resolution, and test detailed hypotheses about the functional roles of these contacts. Finally, we will extend this analysis to a less well- characterized protein-DNA complex (Bam HI endonuclease) to determine the generality of the observed phenomena.